No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Crystal Structure of Tris-inhibited Phospholipase C from Bacillus cereus at 1·9 Å Resolution
Abstract
We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris). The structure has been determined at 1.9 A resolution and refined to R = 20.3%. Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC. The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process. The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+. The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme. This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.
... Maximum activity occurs when the enzyme contains two or three Zn 2+ per mole of protein. In addition, it has been reported that Mg 2+ or Ca 2+ can occupy the metal zinc center during phospholipid hydrolysis [5]. Furthermore, as shown in Figure 8B, PLCBs exhibited a specific activity of around 2000 U/mg in the absence of any trace of ions in the reaction medium. ...
... Maximum activity occurs when the enzyme contains two or three Zn 2+ per mole of protein. In addition, it has been reported that Mg 2+ or Ca 2+ can occupy the metal zinc center during phospholipid hydrolysis [5]. ...
A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLCBs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLCBs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn2+ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLCBs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn2+. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLCBs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLCBs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLCBs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLCBs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.
... Buffers were pre-warmed for at least 20 min before the reaction was initiated by the addition of the PLC. At different time points, 15 μL of the reaction mixture was removed and quenched with 5 μL of 2 M Tris containing 0.4% SDS, pH 8.0 (the Tris cation is an inhibitor of BcPLC [19]). ...
... The high sequence homology between LmPLC and BcPLC allowed us to use the BcPLC crystal structure [13,19,42] to model LmPLC (Fig. 6A). The modeled structure, not surprisingly, is very similar to that for BcPLC showing a helical single domain protein. ...
The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of Listeria monocytogenes.
... Carboxylatobridged zinc complexes are of continuous interest because of their potential relevance as enzyme model [11]. In recent years, there has been a growing interest in enzymes with more than one zinc atom in the active site [12][13][14][15]. Another reason for the importance of zinc in enzyme chemistry is that it can adopt various coordination numbers, i.e., 4, 5, or 6 relatively easily [16,17]. ...
... Calcd. for C 15 (2) A A are identical. The two Zn-O distances involving the bridging carboxylate group 2.018(2), 1.985 (2) A A are also similar and comparable with the Zn-O distance involving the trichloroacetate group 1.993(2) ...
Two novel carboxylato-bridged Zn(II) polymeric complexes [Zn(L)(CCl3COO)]n and [Zn(L)(CF3COO)]n, where [L=2-N-(2′-pyridylimine)benzoic acid] have been synthesized and structurally characterized. The structures of the two complexes are similar with the Zn(L)(Cl3CCO2) or Zn(L)(F3CCO2) units being repeated to form infinite helical chains. In each structure, the neighbouring Zn atoms are bridged sequentially by syn–anti carboxylate groups of the Schiff base.
... Frontiers in Chemistry frontiersin.org concentrations, where it can interact with zincs in the active site (Hansen et al., 1993;Handing et al., 2018). ...
Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N ⁵, N ⁵-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 10⁴ M⁻¹s⁻¹). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 μM; Ki = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).
... Zinc clusters have been observed in the active sites of many enzymes [20] . Seven structures that contain at least two zinc ions have been examined: alkaline phosphatase [21] , phospho- triesterase [22] , Klenow fragment of DNA polymerase I [23] , P1 nuclease [24] , phospholipase C [25] , aminopepti- dase [26] , and leucine aminopeptidase [27] . However, the enzyme from Bacillus cereus functioned reasonably well with only one bound zinc ion [28] . ...
Chlorothalonil hydrolytic dehalogenase (Chd) is one of two reported hydrolytic dehalogenases for halogenated aromatics, and its catalysis is independent of coenzyme A and ATP. Earlier studies have established that the catalytic activity of Chd requires zinc ions. In this study, the metal center of Chd was systematically investigated. The metal content of Chd was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and there were 2.14 equivalents of zinc/mol of protein, indicating that Chd contains a binuclear (Zn²⁺-Zn²⁺) center. It was found that other divalent cations, such as cobalt (Co²⁺) and cadmium (Cd²⁺), could substitute zinc (Zn²⁺) leading to relative activities of 91.6% and 120.0%, whereas manganese (Mn²⁺) and calcium (Ca²⁺) could substitute Zn²⁺ leading to relative activities of 29.1% and 57.0%, respectively. The enzymatic properties of these different metal ion-substituted Chd variants were also compared. Error-prone PCR and DNA shuffling methods were applied to directly evolve Chd to generate variants with higher catalytic efficiencies of chlorothalonil. Enhanced Chd variants were selected based on the formation of clear haloes on Luria-Bertani plates supplemented with chlorothalonil. One variant, Q146R/N168Y/S303G, exhibited a 4.43-fold increase in catalytic efficiency, showing the potential for application in the dehalogenation and detoxification of chlorothalonil contaminated-sites.
... The broadness and low frequency of this band are indicative of hydrogen bonding 15,18-21 . 1 H NMR spectrum of ligand has been recorded in both CDCl 3 and DMSO-d 6 and the results have been given in the experimental section according to alphabeting in Scheme-I. Since the complexes are poorly soluble in CDCl 3 , their NMR spectra were run in DMSO-d 6 . ...
The novel complexes, [CdL2](NO3)2· 2H2O (1) and [Zn(L)(H2O)](NO3)2 (2), have been synthesized by reaction of N, N′-bis(methyl-2-amino-1- cyclopentene-1-dithiocarboxylate)diethylenetriamine (L), with Cd(II) and Zn(II) salts. The ligand and complexes have been characterized by elemental analysis, IR, 1H NMR, 113Cd NMR spectra. Spectroscopic studies show the ligand coordinates to Zn and Cd in tridentate neutral form via three nitrogen atoms. Complex 1 is six-coordinate and complex 2 is four coordinate with one coordinated water molecule. 113Cd NMR confirms a CdN 6 central core for complex 1.
Pioneering work by MacFarlane and Knight showed for the first time that a bacterial toxin — the α-toxin of Clostridium perfringens — also had enzymatic activity as a phospholipase C (PLC; MacFarlane and Knight 1941). This finding stimulated great interest not only in the α-toxin but also in the potentially toxic activity of PLC enzymes produced by other bacteria and in the mechanisms that control the production of these proteins. In addition to the PLCs, bacteria have been shown to produce other phospholipases, such as phospholipase Ds (PLDs) and phospholipase As (PLAs), which differ in the site of phospholipid cleavage (Fig. 1). Today, many phospholipases, produced by both pathogenic and non-pathogenic bacteria, have been described and characterised, but only a few of these enzymes have been shown to be lethal toxins. Nevertheless, some of the enzymes considered to be non-toxic have been shown to play important roles in the pathogenesis of disease. Recent studies have suggested that lethality is a rather crude indicator of the potential roles of these enzymes in disease and that many of these phospholipases appear to exert their effects by allowing the bacteria to colonise the host or by perturbing the metabolism of host cells rather than by directly damaging the host (Titball 1993; Songer 1997). These findings have challenged the conventional criteria by which a bacterial product is considered to be a toxin. The aim of this chapter is to review the broad spectrum of bacterial phospholipases, to discuss the mechanisms by which the expression of phospholipaseencoding genes are regulated, to examine the ways in which these enzymes interact with host cells at a molecular level and to illustrate the ways in which bacterial phospholipases contribute to the pathogenesis of disease.
The PLC class of enzymes has been studied extensively over the past 15-20 years because of their involvement in signaling pathways in which extracellular messages are delivered to the cell to induce a response. Of the PLC isoenzymes, the PI-PLCs have perhaps been examined in the greatest detail because of their key role in initiating cellular response by hydrolyzing the phosphodiester bond of phosphatidylinositols and their phosphorylated derivatives to release the second messengers IP3 and DAG. However, the extended release of DAG that is critical to maintaining the stimulatory response arises from hydrolysis of the more abundant phosphatidylcholine by PC-PLC or by PLD followed by phosphatidic acid phosphatase. Because no eukaryotic PC-PLC has been cloned or isolated in pure form, the phosphatidylcholine-preferring PLC from B. cereus (PLCBc) has emerged as a focal point for investigation and as a putative model for mammalian PC-PLCs. The similarity of the active site of PLCBc with other phosphoryl transfer enzymes has also served as a stimulus for mechanistic studies. The present account details recent studies of this important member of the PLC superfamily of enzymes.
Bone formation and development is a dynamic process involving a series of precisely controlled, distinct, cellular and biochemical events. As bone matures, stage-specific, sequentially expressed bone matrix proteins and hydroxyapatite mineral crystals become incorporated into a collagen fibril-based infrastructure, leading to an organized, stabilized, mineralized matrix. Consequently, this final functional structure contains a variety of matrix proteins present in differential amounts. These include proteins containing: a) glycosaminoglycan (GAG)-bearing proteins - aggrecan, versican, decorin, biglycan, fibromodulin, etc., as well as the free GAG, hyaluronic acid. b) glycoproteins - alkaline phosphatase, osteonectin, tetranectin, thrombospondin (TSP), fibronectin, vitronectin, osteopontin, bone sialoprotein, bone acidic glycoprotein, fibrillin, and tenascin, c) γ-carboxy glutamic acid (Gla)-containing proteins - osteocalcin, and matrix Gla protein (MGP), and d) other proteins - proteolipids, collagen-degrading metalloproteinases and their inhibitors, bone morphogenetic proteins, growth factors, serum-derived proteins, and cell-binding proteins. Emerging new technologies in cell biology, mineral analyses, and transgenic and knockout models are defining the critical proteins and requirements for physiological mineralization.
Introduction Early Studies with Nonbiological Metal Ions Studies of the Cleavage of Phosphate Diesters Promoted by Metal Ion Complexes in Alcohol Fast Metal Ion Promoted Hydrolysis in Wet Alcohol: 20:Zn2II:(RO−) Promoted Hydrolysis of a DNA Model (21b) in Ethanol Conclusions Acknowledgments Abbreviations References
The skeleton is essentially responsible for providing not only structural support and protection to the body's organs, but also for serving as a reservoir for calcium, magnesium, and phosphate ions that are of critical importance in physiology. The fabric of bone is a unique composite of living cells embedded in a remarkable three-dimensional structure of extracellular matrix that is stabilized by a mineral—a carbonate-rich analogue of the geologic mineral hydroxyapatite. During development, mesenchymal cells form the skeleton via two basic pathways. Endochondral bone is formed by an initial condensation of mesenchymal cells that induces the expression of the chondrocytic phenotype. The cartilaginous structure that is formed through the sequential expression of a number of genes that regulate morphogenesis serves as a temporary model. As part of that developmental sequence, the cartilage becomes calcified. The provisional calcified cartilagenous precursor is subsequently replaced by bone. Invasion by blood vessels brings in the cells (osteoclasts) that destroy bone and, in addition, the osteoblastic precursors that will replace the calcified cartilage with bona fide bone. The initial bone formed, the woven bone, is a rather unorganized conglomeration of collagenous and non-collagenous proteins that induce the precipitation of mineral. The mechanical strength of bone is attributable to the presence of mineral, which converts the pliable organic matrix into a more rigid structure. Despite claims of the presence of other mineral phases in bone (for example, brushite, octacalcium phosphate, amorphous calcium phosphate, and whitlockite), current evidence supports the viewpoint that bone mineral is predominantly apatitic, with numerous, perhaps unique, impurities.
The reaction of the polynucleating ligand 1,3,5-tris[bis(2-pyridylmethyl)aminomethyl]benzene (L-TDPA) with Cu(ClO4)(2)center dot 6H(2)O afforded the trinuclear complex [Cu-3(T-TDPA)(H2O)(7)](ClO4)(6)center dot 5H(2)O (I). When the reaction was performed in the presence of the squarate dianion, C4O42-, the compound {[Cu-6(L-TDPA)(2)(mu(1,3)-C4O4)(3)](ClO4)(6)center dot 12H(2)O}(n) (2) was obtained. Both compounds have been characterized by X-ray crystallography, showing a trinuclear and a 1D polymeric structures for 1 and 2, respectively. Complex 2 is composed of unusual hexametallic building blocks. Magnetic susceptibility measurements (SQUID) reveal essentially uncoupled copper(II) ions in complex 1, whereas complex 2 shows weak antiferromagnetic coupling via the squarato bridges within magnetically isolated dicopper(II) subunits of the 1D chain.
Density functional theory (DFT)-Tao-Perdew-Staroverov-Scuseria (TPSS) functional calculations on dizinc complex-mediated phosphodiester cleavage indicate a general base catalytic mechanism. 2-hydroxylpropyl-4-nitrophenyl phosphate (HPNP) favors the bridging of two Zn ions by the formation of two coordination bonds between terminal phosphate oxygens and Zn ions. The Zn-bound hydroxide deprotonates the hydroxyl on the side chain of HPNP and consequently the alkoxide is stabilized by coordination to a Zn ion and a hydrogen-bond to Zn-bound water. A water molecule is tightly bound to two amino protons in the bis(1,4,7-triazacyclononane) ligand and this determines the orientation of HPNP during a nucleophilic attack to form a trigonal bipyramidal PO5 intermediate and it also weakens the bond between phosphorus and the phenolate, which makes the leaving of the latter easier. The phenolate formed after the collapse of the five-coordinated phosphorus intermediate easily coordinates to a Zn ion. Surprisingly, the stabilizing solvent effect for the transition state after the formation of the PO5 intermediate is much stronger (at least 42 kJ·mol-1) than that of all other species as they have solvation energies that fluctuate around 12.6 kJ·mol-1. Thus, the overall free energy barrier for this reaction after reactant-binding and before product release is about 17.0 kJ·mol -1, which is too low to be rate-determining. The rate-determining step is very likely part of the release process of the products. Based on various calculations, we discuss possible reasons for the different catalytic efficiencies of the dizinc complex and the enzymes.
The tetradentate (ONNO) donor Schiff base ligand LH2, derived from the condensation of salicylaldehyde and 1,3-diaminopropane, has been synthesized and reacted with CuBr2 to yield a trinuclear complex with the molecular formula [Cu3L2Br2]. Single crystal X-ray diffraction study reveals that the two terminal copper atoms adopt a square pyramidal geometry, whereas the central copper atom, situated at the inversion centre, is surrounded by four phenoxide oxygen atoms in a square planar fashion. Variable temperature magnetic susceptibility measurement study shows strong antiferromagnetic intra-trimer interactions between the copper centers with a J value of −302 cm−1. EPR study of the complex depicts that, due to the exchange coupling between the copper ions, the EPR spectra were unresolved, having very broad resonances with g = 2.133. A theoretical DFT calculation was also carried out to supplement the experimental results.
Phospholipases are diverse enzymes produced in eukaryotic hosts and their bacterial pathogens. Several pathogen phospholipases have been identified as major virulence factors acting mainly in two different modes: on the one hand, they have the capability to destroy host membranes and on the other hand they are able to manipulate host signaling pathways. Reaction products of bacterial phospholipases may act as secondary messengers within the host and therefore influence inflammatory cascades and cellular processes, such as proliferation, migration, cytoskeletal changes as well as membrane traffic. The lung pathogen and intracellularly replicating bacterium Legionella pneumophila expresses a variety of phospholipases potentially involved in disease-promoting processes. So far, genes encoding 15 phospholipases A, three phospholipases C, and one phospholipase D have been identified. These cell-associated or secreted phospholipases may contribute to intracellular establishment, to egress of the pathogen from the host cell, and to the observed lung pathology. Due to the importance of phospholipase activities for host cell processes, it is conceivable that the pathogen enzymes may mimic or substitute host cell phospholipases to drive processes for the pathogen's benefit. The following chapter summarizes the current knowledge on the L. pneumophila phospholipases, especially their substrate specificity, localization, mode of secretion, and impact on host cells.
The molecular geometry and vibrational frequencies of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II) in the ground state have been calculated by using the Hartree-Fock (HF) and density functional method (B3LYP) with 6-311G(d,p) basis set. The results of the optimized molecular structure are presented and compared with the experimental X-ray diffraction. The energetic and atomic charge behavior of the title compound in solvent media has been examined by applying the Onsager and the polarizable continuum model. To investigate second order nonlinear optical properties of the title compound, the electric dipole (μ), linear polarizability (α) and first-order hyperpolarizability (β) were computed using the density functional B3LYP and CAM-B3LYP methods with the 6-31+G(d) basis set. According to our calculations, the title compound exhibits nonzero (β) value revealing second order NLO behavior. In addition, DFT calculations of the title compound, molecular electrostatic potential (MEP), frontier molecular orbitals, and thermodynamic properties were performed at B3LYP/6-311G(d,p) level of theory.
Synthesis, spectral, biological, and anti-inflammatory investigations of a series of complexes of zinc(II) with 5-(2′-hydroxyphenyl)-3-(4-X-phenyl)pyrazolines of the type (C15H12N2OX)2Zn (where X =–H,–Cl,–CH3,–OCH3) are presented. The complexes were synthesized by reaction of anhydrous zinc(II) chloride with sodium salts of pyrazoline in 1 : 2 molar ratio. Adducts with N and P donor ligands (2,2′-bipyridine, 1,10-phenanthroline and triphenylphosphine) were prepared in 1 : 1 molar ratio. The complexes were characterized by elemental analyses, molecular weight, conductivity, IR, electronic, H, C, P NMR, and FAB mass spectral studies. All complexes are amorphous. Tetrahedral geometry around zinc confirms the presence of two bidentate pyrazoline ligands in zinc(II) 5-(2′-hydroxyphenyl)-3-(4-X-phenyl)pyrazolinates. In adducts pyrazoline is monodentate. Bidentate and monodentate pyrazoline were confirmed by IR, H, C, and P NMR spectral data. All metal complexes were tested for their antibacterial and antifungal activities. Anti-inflammatory activity was also carried out by the carrageenan-induced rat paw edema test. Brine shrimp bioassay was also carried out to study in-vitro cytotoxic properties.
Crystallisation of flexible dipyridyl ligands with either Zn(NO3)2·6H2O or Zn(ClO4)2·6H2O gave co-ordination networks of composition [Zn3(OH)3{(NC5H4)2C3H6}3][NO3]3·8.67H2O 1 [(NC5H4)2C3H6 = 1,3-bis(4-pyridyl)propane]; [Zn2(OH){(NC5H4)2C4H8}3][ClO4]3·H2O·EtOH 2 [(NC5H4)C4H8 = 1,4-bis(4-pyridyl)butane] and [Zn2(OH){(NC5H4)2C7H14}4][ClO4]3·0.5H2O 3 [(NC5H4)2C7H14 = 1,7-bis(4-pyridyl)heptane]. The compounds were characterised by X-ray single crystal diffraction studies. Compound 1 possesses a trinuclear Zn3(OH)3 6-membered ring that acts as a template for the co-ordination framework. Both 2 and 3 possess dinuclear zinc sub-units (Zn2OH) that contain a bridging hydroxyl ion.
Reaction of Zn(ClO4)2·6H2O with pentadentate 1,3-bis(salicylidenamino)propan-2-ol (H2hsalpn) and the tridentate 4-methyl-2,6-bis(morpholinomethyl)phenol (Hbmmp) yielded the trinuclear zinc complex [Zn3(O2CPh)2(hsalpn)2]·2MeOH 1 and the hexanuclear zinc compound [Zn6(OH)6(bmmp)3][ClO4]32. The complexes were characterized by single-crystal X-ray diffraction analyses. Both structures were solved using direct methods and refined on F by full-matrix least squares. Complex 1 consists of one central zinc ion which is octahedrally co-ordinated and two terminal metals surrounded by five donor atoms in the form of a square pyramid. The left and right wing zinc ions are connected to the central metal atom via two phenolate oxygens of the dinucleating ligand and the oxygens of the benzoate ion. The six equivalent tetrahedrally co-ordinated zinc ions in 2 are arranged in form of a regular trigonal prism. Three dinuclear subunits are connected to each other by hydroxide groups. Both compounds display a new type of structure for homotri- and homohexanuclear zinc complexes.
A series of novel luminescent dinuclear zinc(II) diimine complexes with bridging chalcogenolate ligands have been synthesized, in which the two zinc atoms were found to exist in different coordination environment. The luminescence, electrochemical and fluxional behavior of these complexes have been studied. The X-ray crystal structure of [(bpy)Zn2(SC6H4-Cl-p)(μ-SC6H4-Cl-p)(μ-OAc)2] has also been determined.
Five different (pyrazolylborate)zinc hydroxide complexes Tp*Zn−OH (1) were used as hydrolytic reagents towards esters of various acids of phosphorus. Trimethyl phosphate and trimethyl phosphite could not be cleaved. Dimethyl and diphenyl phosphite yielded TptBu,MeZn−OPHO(OR) (2, 3). Triphenyl phosphate reacted slowly producing moderate yields of Tp*Zn−OPO(OPh)2 (4). Tris(p-nitrophenyl) phosphate was cleaved rapidly, forming Tp*Zn−OPO(OC6H4NO2)2 (5) and Tp*Zn−OC6H4NO2 (6). Alkylbis(p-nitrophenyl) phosphates showed intermediate reactivity, losing p-nitrophenolate upon hydrolysis and producing Tp*Zn−OPO(OR)(OC6H4NO2) (7, 8). When phosphorus acid diesters were employed, condensation between the Zn−OH and P−OH functions occurred. This proved to be the convenient way of preparing the organophosphate complexes Tp*Zn−OPO(Ph)2 (9), Tp*Zn−OPO(OPh)2 (4), and Tp*Zn−OPO(OC6H4NO2)2 (5). Six structure determinations showed the structural variability of the resulting complexes.
Numerous studies, both in enzymatic and nonenzymatic catalysis, have been undertaken to understand the way by which metal ions, especially zinc ions, promote the hydrolysis of phosphate ester and amide bonds. Hydrolases containing one metal ion in the active site, termed mononuclear metallohydrolases, such as carboxypeptidase. A and thermolysin were among the first enzymes to have their structures unraveled by X-ray crystallography. In recent years an increasing number of metalloenzymes have been identified that use two or more adjacent metal ions in the catalysis of phosphoryl-transfer reactions (R-OPO3 + R′-OH → R′-OPO3 + R-OH; in the case of the phosphatase reaction R′-OH is a water molecule) and carbonyl-transfer reactions, for example, in peptidases or other amidases. These dinuclear metalloenzymes catalyze a great variety of these reactions, including hydrolytic cleavage of phosphomono-, -di- and -triester bonds, phosphoanhydride bonds as well as of peptide bonds or urea. In addition, the formation of the phosphodiester bond of RNA and DNA by polymerases is catalyzed by a two-metal ion mechanism. A remarkable diversity is also seen in the structures of the active sites of these di- and trinuclear metalloenzymes, even for enzymes that catalyze very similar reactions. The determination of the structure of a substrate, product, stable intermediate, or a reaction coordinate analogue compound bound to an active or inactivated enzyme is a powerful approach to investigate mechanistic details of enzyme action. Such studies have been applied to several of the metalloenzymes reviewed in this article; together with many other biochemical studies they provide a growing body of information on how the two (or more) metal ions cooperate to achieve efficient catalysis.
Sowohl bei enzymatischen als auch bei nichtenzymatischen Katalysen sind zahlreiche Untersuchungen durchgeführt worden, um zu verstehen, wie Metallionen – besonders Zinkionen – die Hydrolyse von Phosphorsäureester- und Amidbindungen unterstützen. Hydrolasen mit einem Metallion im aktiven Zentrum, sogenannte mononucleare Metallohydrolasen, z. B. die Carboxypeptidase A oder Thermolysin, zählen zu den ersten Enzymen, deren Strukturen röntgenographisch aufgeklärt werden konnten. In den letzten Jahren wurden zunehmend mehr Metalloenzyme charakterisiert, in denen zwei oder mehrere benachbarte Metallionen die Katalyse von Phosphoryl- (ROPO3 + R′OH → R′OPO3 + ROH; im Fall der Phosphatasereaktion ist R′-OH ein Wassermolekül) und von Carbonyltransferreaktionen unterstützen, z. B. in Peptidasen und anderen Amidasen. Diese dinuclearen Metalloenzyme katalysieren enorm viele Reaktionen dieser Art: die hydrolytische Spaltung von Phosphorsäuremono-, di- und triesterbindungen, von Phosphorsäureanhydridbindungen sowie die Spaltung von Peptidbindungen oder Harnstoff. Auch die Bildung der Phosphodiesterbindung in RNA und DNA wird von Polymerasen über einen Zwei-Metallionen-Mechanismus katalysiert. Erstaunlich vielfältig sind auch die Strukturen der aktiven Zentren dieser di- oder trinuclearen Metalloenzyme, selbst für Enzyme, die sehr ähnliche Reaktionen katalysieren. Die Strukturbestimmung des aktiven und inaktivierten Enzyms mit gebundenem Substrat oder Produkt, einem stabilen Intermediat oder einem Analogon einer sich im Verlauf der Reaktion bildenden Zwischenstufe ist eine leistungsstarke Methode zur Aufklärung der mechanistischen Details der Enzymkatalyse. Strukturbestimmungen sind für viele der in diesem Artikel beschriebenen Metalloenzyme durchgeführt worden und liefern zusammen mit anderen biochemischen Untersuchungen einen immer besseren Einblick in die Fragestellung, wie die zwei (oder mehr) Metallionen zusammenwirken, um die Reaktionen effizient zu katalysieren.
L. pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine (PC)- phospholipase C (PC-PLC) of Pseudomonas fluorescens. It is unclear, however, whether L. pneumophila exhibits PLC activity. Therefore we screened the L. pneumophila genome for PLClike genes and additionally found lpg0012, which has been shown to encode a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. PlcC expressed in E. coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG) and phosphatidylinositol. Addition of Zn2+ ions activated, while EDTA inhibited, PlcC-derived PLC activity. Protein screening revealed that the three Legionella enzymes and P. fluorescens PCPLC share conserved domains also present in uncharacterized fungal proteins. Seven conserved amino acids were essential to catalysis as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock out mutants indicates Lsp-dependent export of PlcA and likely PlcB, both of which exhibit PG-specific activity and contain a predicted Sec signal sequence. Under the condition of expected impaired type IVB secretion, the Dot/Icm effector PlcC showed cell-associated PC-specific PLC activity. Ectopic expression of PlcC in lung epithelial cells resulted in its accumulation in vesicular structures. A PLC triple mutant, but not single or double mutants, exhibited reduced host cell killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn2+-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.
The bacterial zinc‐metallophospholipases C are produced only by gram‐positive bacteria and are characterised on the basis of the presence of up to three zinc ions in the active site. Some zinc‐metallophospholipases C, like the α‐toxin of Clostridium perfringens, are potent toxins and play key roles in the pathogenesis of disease. Toxicity appears to be related to the ability of the enzyme to interact with phospholipids in host cell membranes and to the hydrolysis of both phosphatidylcholine and sphingomyelin. Significant insight into the mode of action of the zinc‐metallophospholipases has been gained from knowledge of the crystal structures of several members of this group. All of the enzymes possess an enzymatic domain, but only some zinc‐metallophospholipases possess a domain that can play a key role in the recognition of membrane phospholipids. The presence of this domain appears to be necessary for toxicity, but not all enzymes that possess this domain are toxic. Several studies have indicated that membrane active toxins, such as C. perfringens α‐toxin, might be exploited for the treatment of oncogenic disease.
N,N-bis(2-picolyl>glycine (L-H) which was not obtained in the free state was introduced into zinc complexes via its ethyl ester (L-Et) which yielded the intermediate complexes (L-Et)ZnBr2 (1), (L-Et)Zn(NO3)(2) (2), and (L-Et)(2)Zn(ClO4)2 . H2O (3). Autocatalytic hydrolysis in the presence of water turned 2 into [L . Zn(H2O)(2)]NO3 . H2O (4) with an octahedral and 3 into trimeric [L . Zn]ClO4 . H2O (5) with a trigonal-bipyramidal coordination of zinc in the solid state. 5 was found to be a good starting material for the introduction of coligands forming complexes that mimic the coordination of zinc in enzymes with a N,N,O donor set: With imidazole the octahedral complex [L . Zn(Im)(H2O)]ClO4 (6) was obtained, with 2-methylimidazole the trigonal-bipyramidal complex [L . Zn(MeIm)]ClO4 (7), and with diphenyl phosphate the trigonal-bipyramidal complex [L . Zn(Phos)]. 2H(2)O (8). The coordination in the solid state was confirmed for 1 and 4-8 by X-ray work. NMR studies (solid state and solution) and conductivity measurements have revealed various states of dissociation and solvation in solution, with the trigonal-bipyramidal cation [L . Zn(H2O](+) probably being a common and major constituent of all aqueous equilibria.
Treatment of [Zn(Tab)4](PF6)2 (1) with 2,2′-bipyridine (2,2′-bipy), 1,10-phenanthroline (phen), 2,9-dimethyl-1,10-phenanthroline (2,9-dmphen), N-methylimidazole (N-Meim), and 2,6-bis(pyrazol-3-yl)pyridine (bppy) or with CoCl2·6H2O at the presence of N-donor ligands (2,2′-bipy, phen, 4,4′-dimethyl-2,2′-bipyridine (4,4′-dmbpy), 2,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)pyridine (bdmppy)) gave rise to a family of Zn(II) or Co(III)/Co(II) thiolate complexes, [Zn(Tab)2(L)](PF6)2 (2: L = 2,2′-bipy, 3: L = phen, 4: L = 2,9-dmphen), [Zn(Tab)2(N-Meim)2](PF6)2 (5), [Zn(Tab)2(bppy)](PF6)2 (6), [Co(Tab)2(L)2](PF6)3 (7: L = 2,2′-bipy, 8: L = phen, 9: L = 4,4′-dmbpy), and [Co(Tab)(bdmppy)Cl](PF6) (10). These compounds were characterized by elemental analysis, IR spectra, UV−vis spectra, ¹H NMR, electrospray ionization mass spectra and single-crystal X-ray diffraction.
Cocatalytic zinc sites occur in enzymes where two or three metals are closely grouped to bring about catalysis. There are over four dozen representatives of this type of zinc site with the great majority belonging to the Class III hydrolases. A novel feature of these sites is the bridging of two of the metal sites by a side chain moiety of a single amino acid residue, such as the ring nitrogens of the imidazole group of histidine or the carboxylate oxygens of aspartic acid, glutamic acid, or of a carboxylated lysine, LysCO2⁻. Such an interaction requires the metals to be in close proximity to each other. The ligands to cocatalytic zinc sites often come from nearly the entire length of the protein, indicating that the metal sites may be important to the overall fold of the protein as well as to catalytic function. The secondary structure of the protein plays a major role in providing the ligands to these sites. In most cases, ligands are provided by amino acids residing within one or two residues before or after a β-sheet or an α-helix. The zinc ions are often penta-coordinate and arranged in a trigonal bipyramidal geometry. The bridging amino acids and H2O probably have critical roles in catalysis. Their dissociation from either metal atom during catalysis will change the charge on the metal promoting its action as a Lewis acid or allowing interaction with an electronegative atom of the substrate. Thus, it can be envisioned for hydrolytic enzymes that substrate binding involves one zinc site acting as a template for substrate binding, while the other zinc site provides hydroxide for nucleophilic attack on the sp² center of the ester or amide bond of the substrate. In the next step, the roles of the metals can be reversed. In this manner, the metal atoms and their associated ligands play specific roles in each step of the reaction that works to bring about catalysis. The ligands in these sites, in particular the histidines, are often involved in further hydrogen-bonding interactions with other amino acids. These interactions should effect the charge on the metal and the stability of the metal complex, thus fine-tuning catalysis and the stability of the metal sites.
Phosphodiesters are notoriously hydrolytically inert compounds that are demonstrated to have large accelerations of P-OR cleavage promoted by transition and lanthanide metal ions in methanol and ethanol media. This review commentary describes recent findings of how a simple mononuclear and a dinuclear Zn(II) complex promote the cleavage of a series of RNA models and DNA models in alcohol media. The discussion centers on the analysis of the mechanisms of cleavage, energetics of the catalytic process, on recent findings of electrophilic assistance of leaving group departure, and the observation of a rapid hydrolytic reaction of a DNA model promoted by the dinuclear Zn(II) complex in ethanol containing less than 2% water. Copyright © 2009 John Wiley & Sons, Ltd.
This report describes our approach towards modelling the copper cluster active sites of nitrous oxide reductase and the multicopper oxidases/oxygenases. We have synthesized two mesitylene-based trinucleating ligands, MesPY1 and MesPY2, which employ bis(2-picolyl)amine (PY1) and bis(2-pyridylethyl)amine (PY2) tridentate copper chelates, respectively. Addition of cuprous salts to these ligands leads to the isolation of tricopper(I) complexes [(Mes-PY1)Cu(I) (3)(CH(3)CN)(3)](ClO(4))(3)·0.25Et(2)O (1) and [(Mes-PY2)Cu(I) (3)](PF(6))(3) (3) Each of the three copper centers in 1 is most likely four-coordinate, with ligated acetonitrile as the fourth ligand; by contrast, the copper centers in 3 are three-coordinate, as determined by X-ray crystallography The synthesis of [(Mes-PY1)Cu(II) (3)(CH(3)CN)(2)(CH(3)OH)(2)](ClO(4))(6)·(CH(3)OH) (2) was accomplished by addition of three equivalents of the copper(II) salt, Cu(ClO(4))(2)·6H(2)O, to the ligand. The structure of 2 shows that two of the copper centers are tetracoordinate (with MeCN solvent ligation), but have additional weak axial (fifth ligand) interactions with the perchlorate anions; the third copper is unique in that it is coordinated by two MeOH solvent molecules, making it overall five-coordinate. For complexes 2 and 3, one copper ion center is located on the opposite side of the mesitylene plane as the other two. These observations, although in the solid state, must be taken into account for future studies where intramolecular tricopper(I)/O(2) (or other small molecules of interest) interactions in solution are desirable.
The diversity of roles that phospholipases play in biology and medicine is exceptional. In the past decade, this class of enzymes has proven to be considerably more complex than initially perceived and their impact on an assortment of basic cellular processes in eukaryotes, including oncogenesis and inflammation has become widely appreciated. Likewise, there are sundry functions for phospholipases in prokaryotic biology, including their noteworthy contributions to microbial virulence. For example, individual members of a homologous class of phospholipases C (PLCs), usually produced by gram-positive bacteria (GPPLCs), serve vastly different functions in pathogenesis. One member of this class is an extremely potent extracellular toxin (e.g. Clostridium perfringens α toxin), while another contributes to the intricate mechanisms of the intracellular and intercellular trafficking in a facultative intracellular pathogen (e.g. PlcA and PlcB of Listeria monocytogenes).
The P. aeruginosa mutant library appears to be a useful resource both as a source of mutants to analyze gene associations revealed using other
techniques and directly for comprehensive screens for particular phenotypes. Increased automation in such phenotyping should
substantially increase the utility of such screens in the future.
Almost all bacteria require iron for growth and survival. Iron is a constituent of enzymes crucial in oxygen metabolism, electron transfer, and RNA synthesis. Despite its abundance in the earths crust, the availability of iron is severely limited by the very high insolubility of iron(III) at physiological pH. In the presence of oxygen, iron(II) is rapidly oxidized to iron(III) which precipitates as a polymeric oxyhydroxide. The solubility of ferric hydroxide is extremely low (10?38 M) such that, at physiological pH, the concentration of free iron(III) is <10?18 M. This value is far too low for sufficient iron to be acquired by passive diffusion of ions into the cells and to allow growth of aerobic microorganisms. In humans and animals, intracellular iron is mostly bound to ferritin or heme and the extracellular iron found in body fluids is attached to high-affinity iron-binding proteins, such as transferrin found in serum. and lymph and lactoferrin in secretions. The level of free iron in body fluids is therefore in the order of 10?18 M, which is far too low to support the growth of microorganisms. The infected host can therefore be considered as an irondepleted environment. In order to gain access to iron, bacteria have developed several strategies for the acquisition, solubilization, and transportation of iron (Figure 1). One of the most common methods of iron acquisition involves the synthesis and the secretion of low molecular weight iron ligands that are called siderophores2022 After secretion, siderophores chelate iron in the extracellular environment with high affinity (Kd = 10?49 M in some cases) and transport it back into the bacteria. This siderophore-mediated iron acquisition plays an important role in the virulence of pathogenic bacteria19,128 including Pseudomonas102,151,166,167 It has been shown that siderophores are produced by clinical isolates during infection62 and in the case of Pseudomonas aeruginosa in animal models of infection63 In parallel to the iron uptake via siderophores, certain bacteria (many of them pathogens) are able to use heme bound iron from hemoglobin, hemopexin, and haptoglobin58,157,164 At last, some species can acquire iron from transferrins or lactoferrins also involving specific uptake systems in the cell envelope38,139 All iron uptake mechanisms involve a specific outer membrane transporter (OMT) and an ABC transporter for the transport across the outer and the inner membrane, respectively (Figure 1). The energy required is provided by the hydrolysis of ATP for the inner membrane transport and by the proton motive force (pmf) of the inner membrane for the transport through the OMT. In the case of the OMT, the energy is transferred from the inner membrane to the receptor located in the outer membrane by the TonB-ExbB-ExbD complex. The different iron uptake systems of Pseudomonas have recently been reviewed in details120 and will be summarized here. The major objective of this reviewis to address current information about the mechanism of the iron uptake at the molecular level in Pseudomonas and more specifically in P. aeruginosa, the best-studied member of this family of bacteria. The fluorescent properties of the siderophore pyoverdine (Pvd) of this bacterium have been used as a powerful tool to unfold the mechanism of interaction of the ferric-Pvd complex with its OMT FpvA. New insights on the iron uptake in the Burkholderia (formerly Pseudomonas) cepacia complex will be also presented. The different regulators of these iron uptake activities in Pseudomonas have been the topic of a chapter in Pseudomonas Vol III52 and will be just summarized here.
Investigating the molecular archeology of catabolic pathways in pseudomonads and related bacteria and dissecting the different
adaptation bacterial mechanisms has given an extremely detailed picture on the ways how environmentally successful mutants
developed new genetic structures for pollutant metabolism. Principally, the picture is reinforced of extremely effective mechanisms
to capture gene fragments from different sources, recombine them de novo and distribute on whatever mobile conjugative element is at hand.
Type IV pili or fimbriae are non-flagellar, filamentous surface appendages that are associated with a number of biological activities in bacteria. These processes include a form of surface translocation termed twitching motility; bacteriophage sensitivity; attachment to biotic (bacteria, plant, animal) and abiotic surfaces; biofilm development; and the uptake of naked DNA by natural transformation. Many of these biological functions are reliant on the ability of these structures to extend and retract. Type IV pili/fimbriae are found throughout the eubacteria. They are produced by many species of Gram-negative bacteria and have been most extensively studied in Pseudomonas aeruginosa, Neisseria gonorrhoeae, N. meningitidis, Dichelobacter nodosus, Moraxella bovis, Myxococcus xanthus, and Synechocystis species PCC 6803, enteropathogenic Eschericia coli (EPEC) and Vibrio cholerae98,99,143 Type IV pili/fimbriae are also produced by some Gram-positive bacteria including Ruminococcus albus163,168 and probably Streptococcus sanguis100 though evidence for the latter awaits confirmation via molecular analyses. The nomenclature "type IV" is derived from the classification scheme outlined by Ottowin 1975 inwhich he grouped non-flagellar filamentous structures into six types based largely on morphological characteristics156 Interestingly, throughout this nomenclature system Ottow reserved the use of the term pili to specifically refer to conjugative sex pili and used the term "fimbriae" for all other non-flagellar filamentous surface organelles. The term "fimbriae" was also favored by other investigators72,82 however, historically, the terms "pili" and "fimbriae" have been used interchangeably. In reference to the non-flagellar filamentous structures thatwere classified by Ottowas "Group IV," the terminology "type IV pili" and its abbreviation "tfp" have become very much the vogue in recent years and will be adopted in this review. As defined by Ottow (1975) type IV pili are "⋯ flexible, rod-like, polarly inserted fimbriae." The tfp of P. aeruginosa were amongst the first of this class described in bacteria20,21,25,82,104,226 P. aeruginosa tfp have a diameter of 5-6 nm25,50,77,226 and a hollow core of 1.2 nm.77 Due to their retractile nature, the length of these structures varies significantly, but on average are about 1-4 μm in length22,24,27 although they can range in length up to 10 μm25,226 A number of studies have examined tfp production by P. aeruginosa under different culture conditions and have shown that P. aeruginosa tfp are produced throughout plate culture22,119,209,226 in logarithmic broth culture with gentle agitation22,25,226 and by stationary phase bacteria when cultured without aeration or with only gentle agitation22,119,209,226 whereas vigorously shaken stationary phase P. aeruginosa cultures do not produce detectable tfp22,119,209,226 Type IV pili have also been identified in other Pseudomonas species including P. syringae180-183 P. stutzeri84,98 and P. fluorescens138 (see Section 2.6). Historically, however, P. aeruginosa has served as one of the primary model organisms for the investigation of tfp and its associated functions in bacteria. It is for this reason that this chapter will largely focus on the tfp of P. aeruginosa. This chapter will review the current state of knowledge relating to the structure, biogenesis, and function of the tfp of the genus Pseudomonas and will also highlight advances made in the study of these structures in other type IV piliated bacteria.
The PLC class of enzymes has been studied extensively over the past 15–20 years because of their involvement in signaling
pathways in which extracellular messages are delivered to the cell to induce a response. Of the PLC isoenzymes, the PI-PLCs
have perhaps been examined in the greatest detail because of their key role in initiating cellular response by hydrolyzing
the phosphodiester bond of phosphatidylinositols and their phosphorylated derivatives to release the second messengers IP3 and DAG. However, the extended release of DAG that is critical to maintaining the stimulatory response arises from hydrolysis
of the more abundant phosphatidylcholine by PC-PLC or by PLD followed by phosphatidic acid phosphatase. Because no eukaryotic
PC-PLC has been cloned or isolated in pure form, the phosphatidylcholine-preferring PLC from B. cereus (PLCBc) has emerged as a focal point for investigation and as a putative model for mammalian PC-PLCs. The similarity of the active
site of PLCBc with other phosphoryl transfer enzymes has also served as a stimulus for mechanistic studies. The present account details
recent studies of this important member of the PLC superfamily of enzymes.
Aromatic ring hydroxylating dioxygenases play a key role in the biodegradation of numerous environmental pollutants, both
in the natural environment (via natural attenuation) and in the engineered bioremediation systems. Recent structural and mechanistic
information, together with enzyme engineering and strain construction strategies should allow the development of engineered
microorganisms with new and/or optimized degradation abilities. The continued application of these approaches should also
facilitate the development of Rieske non-heme iron dioxygenases with requisite selectivities for specific opportunities in
target direct biocatalysis or metabolic engineering.
The Schiff base ligand (1) and its Zn(II) complex (2) have been synthesized and their crystal structures have been determined. Compound (1) crystallizes in the monoclinic space group C2/c with a=26.993(2), b=5.891(1), β=103.29(1)°, Z=8 and Compound (2) crystallizes in the orthorhombic space group Pbca with a=19.580(3), b=9.416(2), Z=8 and In the crystal structure of the free Schiff base ligand (1), the existence of a strong intramolecular N–H⋯O hydrogen bond [] is observed. The C–N amine bond and C–N–C bond angle are 1.345(4) Å and 124.4(3)°, respectively. The C3O1 and C4C5 bond lengths [1.274(4) and 1.351(5) Å] are shortened by the pronounced quinoidal effect. In solution, compound (1) is in tautomeric equilibria (phenol–imine, O–H⋯N keto-amine, O⋯H–N forms), as supported by 1H NMR and UV–visible data. In the crystal structure of the Zn(II) complex (2), zinc atom has a distorted tetrahedral coordination. One of the pyridine N atom of the ligands is in close contact with the Zn(II) atom []. It is interesting that the C–N amine bond [1.345(4) Å] in compound (1) changes to the imine bond [1.27(5) Å] in the Zn(II) complex (2).
1,2-sn-Diacylglycerol (DAG) is a family of lipidic molecular species varying in the lengths and desaturation levels of acyl groups esterified at positions sn-1 and sn-2 of the glycerol backbone. In plant cells, DAG originating from plastid and from extraplastidial membranes have distinct molecular signatures, C18/C16 and C18/C18 structures, respectively. Under normal conditions, DAG is consumed nearly as fast as it is produced and is therefore a transient compound in the cell. In plants, DAG proved to be the most basic ingredient for cell membrane biogenesis and fat storage, but we still lack formal evidence to assert that DAG is also an intracellular messenger, as demonstrated for animals. From the biochemical and molecular comparisons of the best known DAG-manipulating proteins of prokaryotic and eukaryotic cells (phosphatidate phosphatases, diacylglycerol kinases, MGDG synthase, protein kinase C, etc.) this review aims to identify general rules driving DAG metabolism, and emphasizes its unique features in plant cells. DAG metabolism is an intricate network of local productions and utilizations: many isoenzymes can catalyse similar DAG modifications in distinct cell compartments or physiological processes. The enzymatic- or binding-specificity for DAG molecular species demonstrates that discrete DAG molecular subspecies fluxes are finely controlled (particularly for C18/C16 and C18/C18 structures in plastid membrane biogenesis). Eventually, this review stresses the diversity of structures and functioning of DAG-manipulating proteins. As a consequence, because DAG metabolism in plants is unique, the deciphering of genomic information cannot rely on homology searches using known prokaryotic, animal or yeast sequences, but requires sustained efforts in biochemical and molecular characterizations of plant DAG-manipulating proteins.
In this study we compare the binding energies of polycoordinated complexes of Zn2+ within cavities composed of model “hard” (H2O, OH−) or “soft” (CH3SH, CH3S−) ligands. Ab initio supermolecule computations are performed at the HF and MP2 levels using extended basis sets to determine the binding energies and their components as a function of: the number of ligands, ranging from three to six; the net charge of the cavity; and the “hard” versus “soft” character of the ligands. These ab initio computations are used to test the reliability of the SIBFA molecular mechanics procedure, originally formulated and calibrated on the basis of ab initio computations, for such charged systems. The SIBFA intermolecular interaction energies match the corresponding ab initio values using a coreless effective potential split-valence basis set with a relative error of ≤3%. Extensions to binuclear Zn2+ complexes, such as those that occur in the Zn-binding sites of Gal4 and β-lactamase proteins, are performed to test the applicability of the methodology for such systems. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1011–1039, 2000
To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus (PLCBc) might play in binding and catalysis, selected mutants were prepared through site-directed mutagenesis of the plc gene. The mutants were then expressed in Escherichia coli and purified as fusion proteins with the maltose binding protein (MBP). Kinetic analysis showed that mutations at Glu4 had only modest effects on the catalytic activity, whereas those at Asp55 led to proteins whose values for kcat/KM were 10(4)-10(6) times less than that of the wild-type enzyme. The modest decrease in catalytic activity and the pH-dependent profile of the E4L mutant strongly suggest that glutamic acid at position 4 is not the general base in the PLCBc-catalyzed reaction. Rather, the results support the hypothesis that Glu4 is primarily involved in substrate binding, perhaps by electrostatic stabilization of the positive charge of the choline moiety of the phosphatidylcholine substrate. Examination of X-ray crystallographic data of PLCBc and its various complexes reveals that the carboxylate side chain of Asp55 is positioned such that it could activate a water for nucleophilic attack on the substrate or serve as a ligand for Zn1. However, the involvement of the side chain of Asp55 as an important Zn1 ligand is not consistent with the atomic absorption and thermostability data obtained for the D55L mutant, which are virtually identical with that of the wild-type enzyme. The large reduction in the measured kcat/KM of the D55E, D55N, and D55L mutants of PLCBc indicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLCBc.
The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.
To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(II) complex (4) at (s)spH 9.8 and 25 degrees C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k(obs)(6a) = (1.47 +/- 0.09) x 10(-4) s(-1) and k(obs)(6b) = (2.08 +/- 0.09) x 10(-6) s(-1), respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, Kb, and the catalytic rate constant, kcat(max), for the unimolecular decomposition of the 8:4 Michaelis complex. A Brønsted plot of the log (k(cat)(max)) vs. sspKa(phenol) (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log kcat(max) = (-0.80 +/- 0.07)(s)spKa + (10.2 +/- 1.0) which includes the datum for 6a. The datum for 6b, which reacts approximately 70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4.
Phosphatidylcholine-preferring phospholipase C is a trinuclear zinc-dependent phosphodiesterase, catalyzing the hydrolysis of choline phospholipids. In the present study, density functional theory is used to investigate the reaction mechanism of this enzyme. Two possible mechanistic scenarios were considered with a model of the active site designed on the basis of the high resolution X-ray crystal structure of the native enzyme. The calculations show that a Zn1 and Zn3 bridging hydroxide rather than a Zn1 coordinated water molecule performs the nucleophilic attack on the phosphorus center. Simultaneously, Zn2 activates a water molecule to protonate the leaving group. In the following step, the newly generated Zn2 bound hydroxide makes the reverse attack, resulting in the regeneration of the bridging hydroxide. The first step is calculated to be rate-limiting with a barrier of 17.3 kcal/mol, in good agreement with experimental kinetic studies. The zinc ions are suggested to orient the substrate for nucleophilic attack and provide electrostatic stabilization to the dianionic penta-coordinated trigonal bipyramidal transition states, thereby lowering the barrier.
Thesis (Ph. D.)--University of Texas at Austin, 2005. Supervisor: Stephen F. Martin. Vita. Includes bibliographical references. Requires PDF file reader.
In der vorliegenden Arbeit sollte die Frage geklärt werden, ob die atoxischen gentechnisch hergestellten Alphatoxinvarianten (rAT) 121A/91 und 121A/91-His212 die zur Immunisierung gegen das C. perfringens Alphatoxin Verwendung finden können. Das rAT121A/91 ist das rekombinante Analog der atoxischen Alphatoxinvariante 121A/91, die vom natürlich vorkommenden C. perfringens Feldisolat 121A/91 sezerniert wird. Die Variante 121A/91-His212 ist durch in vitro Mutagenese aus dem AT121A/91 hervorgegangen. Hierbei wurde das Arginin an Position 212, entsprechend der Wildtyptoxin-Sequenz, durch ein Histidin ersetzt. Studien mit den beiden rekombinanten Varianten und dem Alphatoxinspezifischen monoklonalen Antikörper 3B4 zeigten, daß die Alphatoxinvariante 121A/91- His212 eine höhere Bindungsaktivität gegenüber dem monoklonalen Antikörper aufweist als das rAT121A/91. Dies läßt darauf schließen, daß sich das His212 in einem für die Bindung essentiellen Aminosäurebereich befindet.
Die beiden Alphatoxinvarianten ließen sich effektiv in E. coli pASK-IBA2-Expressionsvektoren exprimieren und affinitätschromatographisch aus dem Periplasmaextrakt aufreinigen. Die Alphatoxinvarianten waren bei Immunisierungsversuchen in der Maus gut verträglich. Bei der Verwendung der Impfkandidaten in MF59, einem Öl in Wasser Adjuvans, traten bei der s.c. Immunisierung keine und bei der i.p. Applikation nur geringgradige Allgemeinstörungen auf. Die s.c. Vakzinierung mit dem durch in vitro Mutagenese gewonnenen rAT121/91-His212 in Alu-Gel führte hingegen zu entzündlichen Veränderungen an der Applikationsstelle.
Im ELISA konnte gezeigt werden, daß die i.p. Immunisierung mit beiden Varianten in MF59 zu Alphatoxin-spezifischen Antikörper Titern von 1: 1.024.000 führte. Obwohl sich die Titer nicht in ihrer Höhe unterschieden, konnte im in vitro Hämolyse-Neutralisationsassay nachgwiesen werden, daß die mit dem rAT121A/91-His212 induzierten Antiseren eine stärkere antihämolytische Wirkung aufwiesen. Da die intraperitoneale Applikation auf die Anwendung im Mausmodell begrenzt ist, wurde das rAT121A/91-His212 zudem bei subcutaner Immunisierung getestet. In Kombination mit MF59 führte dies zu vierfach geringeren Antikörperkonzentrationen als nach intraperitonealer Immunisierung. Trotz niedrigerer Alphatoxin-erkennender Antikörpertiter war der Anteil antihämolysierender Antikörper jedoch um den Faktor zwei höher, als nach entsprechender i.p. Immunisierung.
Die enzymatische Aktivität des Alphatoxins konnte mit beiden Seren in Kombination mit MF59 unabhängig vom Applikationsweg neutralisiert werden. Die Seren, die mit Alu-Gel als Adjuvans bzw. ohne die Zugabe eines Adjuvans gewonnen worden waren, besaßen keine PLC-inhibierende Wirkung.
Im Toxinchallenge-Versuch war das rAT121A/91-His212 dem rAT121A/91 eindeutig überlegen. Bei der Verwendung von rAT121A/91-His212 in Kombination mit MF59 überlebten bei i.p. Immunisierung bis zu 76% und bei s.c. Applikation bis zu 42% der Tiere die Belastung mit dem Wildtyptoxin. Bei den Tieren der Adjuvanskontrollen betrug die maximale Überlebensrate 17%. Die i.p. Immunisierung mit rAT121A/91 in MF59 führte hingegen nicht zu einer nachweisbaren Schutz der Tiere.
Die im in vivo Belastungsversuch gewonnenen Ergebnisse wurden in einem in vitro Neutralisationsversuch bestätigt. Hierbei wurde mit Antiserum präinkubiertes Alphatoxin Mäusen intraperitoneal appliziert. Nur Tiere, denen ein Gemisch aus Alphatoxin mit durch i.p. Immunisierung von rAT121A/91-His212 in MF59 gewonnem Serum injiziert wurde, überlebten.
Nach Vakzinierung von Mäusen mit dem durch in vitro Mutagenese gewonnenen rAT121A/91-His212 gelang es, Antiseren zu gewinnen, die sowohl die enzymatische Aktivität, als auch die hämolytische Wirkung des Alphatoxins neutralisierten. Die intraperitoneale Vakzinierung mit der Alphatoxinvariante und einem Öl in Wasser Adjuvans war zudem geeignet, die letale Wirkung des Wildtyptoxins sowohl im in vitro Neutralisationsassay als auch im in vivo Belastungsversuch vollständig zu inhibieren bzw. signifikant zu reduzieren. Das rAT121A/91-His212 ist somit ein Impfantigen, das sich für eine aktive Immunisierung gegen das Alphatoxin von C. perfringens eignet. The goal of the present study was to determine the suitability of two non-toxic recombinant Clostridium perfringens alphatoxin variants (rAT), 121A/91 and 121A/91-His212, for use as potential vaccines. The rAT121A/91 is the recombinant analogue of the naturally occurring, non-toxic variant 121/91A. The 121A/91-His212 was constructed from 121/A91 using in vitro mutagenesis. The Arg at position 212 was substituted for a His residue to mimic the toxin sequence of the wild type toxic alphatoxin. Studies examining the binding of the alphatoxin-specific monoclonal antibody 3B4 indicated more mab 3B4 bound to the 121A/91-His212 protein than the 121A/91 protein. This indicates that the His 212 residue is located in a region which is essential for mab 3B4 binding. Both variant alphatoxins could be effectively expressed using E. coli pASK-IBA2-expression vectors and were purified from the periplasmic extract.
Immunisations with the alphatoxin variants were well tolerated by mice. Combined with the oil-in-water emulsion MF59, the IP immunisation resulted in a slight reaction measured by change in the condition of the mice and no change was seen with the SC immunisation. The rAT121A/91-His212 was also combined with aluminum gel and given by the SC route. This immunisation resulted in an inflammatory reaction at the injection site.
Sera from mice immunised IP with either of the variant alphatoxins in MF59 were tested in an ELISA and in an in vitro haemolysis neutralisation assay. This route of immunisation induced ELISA anti-alphatoxin titres of 1:1,024,000. Although the magnitude of the ELISA titre was the same for both variants, the rAT121A/91-His212 variant induced anti-sera with higher anti-haemolytic activity. As the IP immunisation is restricted to the mouse model, SC vaccination was also evaluated. Using this route with rAT121A/91-His212 and MF59 resulted in four-fold lower ELISA titres than induced by the IP immunisation. However, the antihaemolytic activity of the sera induced by the SC immunisation were twice the magnitude of the IP immunisation.
Sera from the immunised mice were also tested for the ability to inhibit the enzymatic cleavage of alphatoxin substrates. Sera from mice immunised with either variant and MF59 were able to inhibit this reaction, however sera from aluminum gel adjuvanted vaccine groups or non-adjuvanted groups did not inhibit PLC-hydrolysis.
In toxin challenge assays the rAT121A/91-His212 was clearly superior to the rAT121A/91. When the animals were vaccinated using the MF59 adjuvant, survival rates for the rAT121A/91-His212 variant were 76% for the IP vaccinated group and 42% for the SC vaccinated group. Immunisation with the rAT121A/91 variant in MF59 did not produce any enhancement of survival when compared to the adjuvant controls. In these studies the highest survival rate in the adjuvant controls was 17%. The results from the in vivo challenge studies were verified by an in vitro neutralisation assay. In this assay alphatoxin is pre-incubated with antiserum prior to injection into mice. The results of this study showed that only animals that were injected with alphatoxin, which had been pre-incubated with anti-sera from rAT121A/91-His212 and MF59 immunised animals, survived. After the vaccination of mice with the construct rAT121A/91-His212, which was obtained through in vitro mutagenesis, anti-sera that neutralised the enzymatic activity and the haemolytic activity of C. perfringens alphatoxin were induced. The intraperitoneal immunisation using this construct and an oil-in-water adjuvant inhibited the lethal effects of the wild-type toxin in in vitro neutralisation assays and significantly reduced it in vivo challenge assays. This shows that the rAT121A/91-His212 variant can be used for the active immunisation against the alphatoxin of C. perfringens.
The assembly formation of Zn cluster compounds comprising pyridinealcohol ligands is described and their solution and solid state features have been determined using NMR spectroscopy and X-ray diffraction. The di-Zn complex 5 and tetra-Zn complex 7 may serve as accessible structural models for the active site of various multinuclear Zn-containing metalloenzymes. Their phosphoester cleavage activity was tested and correlated with their dynamic structural features.
An analysis of the geometry and the orientation of metal ions bound to histidine residues in proteins is presented. Cations are found to lie in the imidazole plane along the lone pair on the nitrogen atom. Out of the two tautomeric forms of the imidazole ring, the NE2-protonated form is normally preferred. However, when bound to a metal ion the ND1-protonated form is predominant and NE2 is the ligand atom. When the metal coordination is through ND1, steric interactions shift the side chain torsional angle, chi 2 from its preferred value of 90 or 270 degrees. The orientation of histidine residues is usually stabilized through hydrogen bonding; ND1-protonated form of a helical residue can form a hydrogen bond with the carbonyl oxygen atom in the preceding turn of the helix. A considerable number of ligands are found in helices and beta-sheets. A helical residue bound to a heme group is usually found near the C-terminus of the helix. Two ligand groups four residues apart in a helix, or two residues apart in a beta-strand are used in many proteins to bind metal ions.
An analysis of the geometry of metal binding by carboxylic and carboxamide groups in proteins is presented. Most of the ligands are from aspartic and glutamic acid side chains. Water molecules bound to carboxylate anions are known to interact with oxygen lone-pairs. However, metal ions are also found to approach the carboxylate group along the C-O direction. More metal ions are found to be along the syn than the anti lone-pair direction. This seems to be the result of the stability of the five-membered ring that is formed by the carboxylate anion hydrogen bonded to a ligand water molecule and the metal ion in the syn position. Ligand residues are usually from the helix, turn or regions with no regular secondary structure. Because of the steric interactions associated with bringing all the ligands around a metal center, a calcium ion can bind only near the ends of a helix; a metal, like zinc, with a low coordination number, can bind anywhere in the helix. Based on the analysis of the positions of water molecules in the metal coordination sphere, the sequence of the EF hand (a calcium-binding structure) is discussed.
An extension of Furnas's method is described. The variation of intensity of an axial reflection as the crystal is rotated about the goniometer axis is used to give a curve of relative transmission T against azimuthal angle ϕ for the corresponding reciprocal lattice level. Transmission coefficients for any general reflexion hkl are then given approximately by T(hkl) = [T(ϕinc) + T(ϕret)]/2 where ϕinc and ϕret are the azimuthal angles of the incident and reflected beams. Equations are derived for (ϕinc and ϕret and the accuracy of the method is discussed.
A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.
Crystals of phospholipase C from Bacillus cereus have been grown and are suitable for X-ray diffraction analysis to at least 2.2 Å. The crystals belong to the tetragonal space group P41 (or P43); unit cell dimensions are . The unit cell appears to contain 57% solvent and the asymmetric unit one molecule of enzyme.
1. The zinc content and metal ion dependence of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus have been examined. 2. The native enzyme contained about 2 atoms of tightly bound zinc/molecule. 3. Incubation of the enzyme with EDTA or with o-phenanthroline caused inactivation. The inactivation was accompanied by the removal of one zinc atom from the enzyme and could be fully reversed by the addition of Zn2+ or Co2+ to the enzyme and partly reversed by Mn2+ or Mg2+. 4. Prolonged exposure to o-phenanthroline removed the second zinc atom also and produced an enzyme species which was reactivated by Zn2+ only. Full reactivation was accompanied by the binding of about two zinc atoms to the enzyme. 5. The results are consistent with the view that phospholipase C is a zinc metalloenzyme.
Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.
Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.
The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.
Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.
The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites.
The structure of the water-soluble bacteriochlorophyll a protein (Bchl protein) from the green photosynthetic bacterium Prosthecochloris aestuarii has been refined at 1.9 A resolution to a crystallographic residual of 18.9%. The refinement was carried out without knowledge of the amino acid sequence and has led to an "X-ray sequence". The structure consists of seven Bchl molecules enclosed within a protein "bag" and the refinement supports the general conformation of the molecule described previously. The refinement also supports the previous suggestion that the ligands to the seven Bchl magnesiums are, respectively, five histidines, a carbonyl oxygen from the polypeptide backbone of the protein, and a bound water molecule. The conformations of the seven Bchl head-groups are described in detail. In two cases the magnesium atoms are approximately 0.48 A "below" the plane of the conjugated macrocycle while in the other five cases the atoms are, on average, 0.48 A "above" the plane. The acetyl ring substituents are more-or-less coplanar with the dihydrophorbin macrocycle, consistent with a previous resonance Raman study. The conjugated atoms in each of the seven macrocycles have significant departures from strict planarity. These deviations are similar for Bchls 1, 2 and 3 (class I) and are also similar for Bchls 4, 5, 6 and 7 (class II). Ethylchlorophillide also belongs to class II. The out-of-plane deformations for the class I and class II bacteriochlorophylls appear to correspond to two distinct modes of bending or curvature of the dihydrophorbin macrocycle.
The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 Å resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 Å was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 Å data and extending in steps to 2.8 Å resolution, proceeded to a final overall crystallographic R factor of 0.256.
A simplified method for the purification of phospholipase C from Bacillus cereus is described. The enzyme is homogeneous in disc electrophoresis and in dodecylsulphate-polyacrylamide gel electrophoresis as well as in the analytical ultracentrifuge. The enzyme had a molecular weight of 23000, pI= 6.5 and consisted of one subunit. A divalent metal ion was necessary, Zn2+ was the most active. The activation of factor VII by tissue thromboplastin was reversible when tissue thromboplastin was destroyed by phospholipase C.
Phospholipase C (EC 3.1.4.3) from Bacillus cerens was found to be inactivated by ethylenediaminetetraacetate and o-phenanthroline. The inactivation was both time and concentration dependent and was reversed by the addition of Zn2+ and to a lesser extent Mn2+. Other cations including Ca2+ did not reverse the inhibition. Extensive dialysis inactivated the enzyme, and this inactivation could be reversed by the addition of thiol-containing compounds. The ratio of Zn2+ to protein increased with purification of the enzyme, and spectra of o-phenanthroline-treated enzyme showed Zn2+ bound to the protein. The evidence would indicate this phospholipase is a metalloenzyme requiring Zn2+ and thiol groups for activity ; this is in contrast with the classical idea of Ca2+ requirement for phospholipase-C activity.
A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.